D-lactate dehydrogenase, engineered strain containing D-lactate dehydrogenase and construction method and use of engineered strain

ABSTRACT

The present invention provides D-lactate dehydrogenase, an engineered strain containing the D-lactate dehydrogenase and a construction method and use of the engineered strain. The present invention discloses a D-lactate dehydrogenase which has unique properties and is from Thermodesulfatator indicus, and the D-lactate dehydrogenase has good thermophily and heat stability. By using the D-lactate dehydrogenase and the gene engineering reconstruction method, a fermentation product of the reconstructed Bacillus licheniformis can be redirected to optically-pure D-lactic acid with a high yield from naturally produced 2,3-butanediol, and the optical purity of the produced D-lactic acid reaches 99.9%; and raw materials for fermentation are low-cost, and a fermentation state is between an anaerobic fermentation state and a microaerobic fermentation state. By using the inventive method for producing D-lactic acid through fermentation at high temperature, the production cost can be reduced, the production efficiency can be improved and there is a wide industrial application prospect for the inventive method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/073,051, filed on Jan. 25, 2017, which is the U.S. national phase of International Application No. PCT/CN2017/072647 filed Jan. 25, 2017, and claims priority to Chinese Patent Application No. 201610056883.7 filed Jan. 27, 2016, the disclosures of each of which are hereby incorporated by reference in their entireties.

The Sequence Listing associated with this application is filed in electronic format via EFS-Web and is hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 1805282_ST25.txt. The size of the text file is 50,426 bytes, and the text file was created on Jul. 25, 2018.

FIELD OF THE INVENTION

The present invention relates to the field of gene engineering, and in particular to a D-lactate dehydrogenase, a construction method of a bacillus gene engineering strain containing the D-lactate dehydrogenase and use of the engineered strain in production of D-lactic acid.

DESCRIPTION OF THE PRIOR ART

Lactic acid is an important industrial raw material and can be applied to pharmaceutical, food, cosmetic and petrochemical industries, etc. In recent years, as a monomer, lactic acid is also used for synthesizing high-strength biodegradable plastic polylactic acid (PLA). Traditionally, PLA is polymerized by using highly optically-pure L-lactic acid.

Furthermore, by using mixed L-lactic acid and D-lactic acid, the mechanical performance, heat stability and hydrolysis resistance of products using polylactic acid as materials can be obviously improved, thereby greatly exciting the market demands for D-lactic. Although lactic acid can be synthesized by using petroleum as a raw material, the synthesized lactic acid is a mixture of two types of isomerides and is not applicable to production of PLA. Highly optically-pure L-lactic acid and D-lactic acid needed for producing PLA can only be produced through microbial fermentation, and thus many scholars carry out researches on production of D-lactic acid by using a microbial fermentation method.

High-temperature fermentation can minimize the risk of pollution, improve the conversion rate of raw materials and reduce heat supply cost. Up to now, production of optically-pure L-lactic acid at high temperature has been widely studied, which makes a huge contribution to the commercialized production of L-lactic acid. In regard to D-lactic acid, people always eager to realize high-temperature fermentation production of D-lactic acid. However, there are few reports about thermophilic production of D-lactic acid, and the concentration, production speed and rich culture medium components for reported D-lactic acid are not suitable for industrial production. Therefore, it is very necessary to develop a low-cost strong microbial platform to produce D-lactic acid at high temperature.

D-lactate dehydrogenase on which nicotinamide adenine dinucleotide (NADH) depends is a key enzyme for microbial synthesis of D-lactic acid. However, most natural D-lactate dehydrogenases are not stable to heat, and this is one of major bottlenecks for microbial production of D-lactic acid at high temperature. It is reported that, as proved by in-vitro determination of enzyme activity of D-lactate dehydrogenases in Lactobacillus plantarum, this enzyme has very high enzyme activity at 42° C., but is fully deactivated after incubation for 3 min at 50° C., while L-lactate dehydrogenases in Lactobacillus plantarum after treatment by using the same process still reserve 94% of enzyme activity. As also shown by other in-vivo experiments, D-lactate dehydrogenases are easily deactivated under a high-temperature condition. On the whole, the low activity or extremely poor heat stability of the D-lactate dehydrogenases reported above prevents the production of D-lactic acid under a high-temperature condition by using the D-lactate dehydrogenases. In order to realize high-efficiency production of D-lactic acid at high temperature, it is an important task to find a D-lactate dehydrogenase having high heat stability.

Heat-resistant Bacillus licheniformis ATCC 14580 is a facultative anaerobic, Gram-positive endospore-type bacterium. For a microbial fermentation platform, it has a lot of potential advantages as follows: 1) it can use various pentoses and hexoses; 2) it has a fast cell growth speed and thus can shorten the fermentation period; 3) it can be subjected to genetic operations; and 4) it is a “Generally Recognized as Safe” strain generally accepted by the Food and Drug Administration. These advantages show that strain ATCC 14580 can be used as an ideal platform strain. Up to now, this strain has already been proved to be usable for high-temperature fermentation production of 2,3-butanediol.

Therefore, one skilled in the art devotes himself to developing an engineered strain for D-lactic acid with a high yield at high temperature and preparation and use thereof, by using heat-resistant Bacillus licheniformis ATCC 14580 as an original strain.

SUMMARY OF THE INVENTION

With regard to the defects that most natural D-lactate dehydrogenases in the prior art are poor in heat stability, the cost of the existing thermophilic production of D-lactic acid is high and the concentration and production speed of D-lactic acid are not suitable for industrial production, the present invention provides an engineered strain for producing D-lactic acid with a high yield at high temperature, and preparation method and use of the strain. The present invention discloses a D-lactate dehydrogenase which has unique properties and is extracted from Thermodesulfatator indicus, and this enzyme has extremely good thermophily and heat stability. After performing codon optimization for the enzyme via colibacillus, by using the optimized enzyme and the gene engineering construction method, a fermentation product of the reconstructed Bacillus licheniformis can be redirected to optically-pure D-lactic acid with a high yield from naturally produced 2,3-butanediol.

In one aspect, the present invention provides a D-lactate dehydrogenase. In one specific implementation mode, the D-lactate dehydrogenase has one of the following amino acid sequences:

1) an amino acid sequence expressed by SEQ ID No. 1;

2) an amino acid sequence derivatively produced from the amino acid sequence expressed by SEQ ID No. 1 through substitution, deletion, insertion or addition of one or more amino acid residues, and a protein produced by the amino acid sequence having D-lactate dehydrogenase activity;

3) an amino acid sequence produced from the amino acid sequence expressed by SEQ ID No. 1 through conservative replacement; and

4) an amino acid sequence having at least 80% of homology with the amino acid sequence expressed by SEQ ID No. 1.

Further, the substitution, deletion, insertion or addition of the amino acid residues occurs outside a functional domain comprising enzyme catalytic sites, ligand binding sites and NAD binding sites of the D-lactate dehydrogenase.

Further, according to relative positions, key amino acids at the enzyme catalytic sites are Arg at position 229, Glu at position 258 and His at position 290; key amino acids at the ligand binding sites are Ser, Ala and Gly positions 75-77, Tyr at position 99, Arg at position 229, His at position 290 and Phe at position 293; and key amino acids at the NAD binding sites are Tyr at position 99, Ile at position 104, Gly at position 150, Gly, Lys, Ile and Gly at positions 152-155, Tyr, Asp and Pro at positions 172-174, His, Cys, Pro and Leu at positions 199-202, Asn at position 206, Met at position 209, Thr, Ala and Arg at positions 227-229, Asp and Val at positions 253 and 254, His at position 290, and Ala and Phe at positions 292 and 293.

In another aspect, the present invention provides a nucleotide sequence. In one specific implementation mode, the nucleotide sequence codes the D-lactate dehydrogenase according to claim 1.

Further, the nucleotide sequence is one of the following nucleotide sequences:

1) a nucleotide sequence expressed by SEQ ID No. 2;

2) a nucleotide sequence expressed by SEQ ID No. 3;

3) a nucleotide sequence having more than 80% of homology with the nucleotide sequence expressed by SEQ ID No. 2 or SEQ ID No. 3; and

4) a nucleotide sequence hybridized with a complementary chain of the nucleotide sequence expressed by SEQ ID No. 2 or SEQ ID No. 3 under a high-stringency condition.

In another aspect, the present invention provides use of the D-lactate dehydrogenase as described above or the nucleotide sequence as described above in production of D-lactic acid.

In another aspect, the present invention provides a gene engineering strain. In one specific implementation mode, the gene engineering strain has the D-lactate dehydrogenase as described above or a D-lactate dehydrogenase coded by the nucleotide sequence as described above.

Further, in the gene engineering strain, a pathway for synthesizing L-lactic acid by pyruvic acid is blocked and a pathway for synthesizing 2,3-butanediol by pyruvic acid is blocked.

Preferably, the pathway for synthesizing L-lactic acid by pyruvic acid is blocked by deactivating or deleting an L-lactate dehydrogenase gene; and the pathway for synthesizing 2,3-butanediol by pyruvic acid is blocked by deactivating or deleting one or both of an acetolactate synthase gene and an acetolactate decarboxylase gene.

Preferably, in the gene engineering strain, one or more of a pathway for synthesizing formic acid by pyruvic acid, a pathway for synthesizing acetic acid by pyruvic acid, and a pathway for synthesizing ethanol by pyruvic acid are blocked.

Preferably, the pathway for synthesizing formic acid by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate formate-lyase gene and a pyruvate formate-lyase activating enzyme gene; the pathway for synthesizing acetic acid by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate dehydrogenase gene and an acetokinase gene; and the pathway for synthesizing ethanol by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate dehydrogenase gene and an ethanol dehydrogenase gene.

Preferably, an original D-lactate dehydrogenase gene in the gene engineering strain is deactivated or deleted.

Further, an original strain of the gene engineering strain is thermophilus.

Further, an original strain of the gene engineering strain is bacillus.

Preferably, the bacillus is selected from a group consisting of Bacillus licheniformis, Bacillus coagulans, B. subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus circulans and Aneurinibacillus aneurinilyticus.

Preferably, a restrictive modification system in the original strain is deactivated or knocked out.

Preferably, the original strain is a ΔhsdR1ΔhsdR2 double-mutant strain MW3 of Bacillus licheniformis ATCC 14580.

Further, a promoter of a coding gene of the above D-lactate dehydrogenase is reconstructed into a constitutive promoter.

Preferably, the constitutive promoter is a promoter P_(als) of α-acetolactate synthase of Bacillus licheniformis ATCC 14580, a P_(c) promoter in plasmid pMMPc, a P₄₃ promoter in B. subtilis or a promoter P_(ldh) of L-lactate dehydrogenase of Bacillus licheniformis ATCC 14580.

More preferably, the constitutive promoter is a promoter P_(als) of α-acetolactate synthase of Bacillus licheniformis ATCC 14580.

Further, the gene engineering strain is Bacillus licheniformis BN11, the preservation number of which is CCTCC NO: M2016026 and which was preserved in China Center for Type Culture Collection on Jan. 8, 2016. Bacillus licheniformis BN11 was deposited on Jan. 8, 2016 under the Budapest Treaty with the China Center for Type Culture Collection at Wuhan University, Wuhan 430072, P.R. China, under accession CCTCC M 2016026.

In another aspect, the present invention provides a method for preparing the gene engineering strain as described above. In one specific implementation mode, the method comprises: deactivating or deleting an L-lactate dehydrogenase gene in an original strain; and introducing the nucleotide sequence of the D-lactate dehydrogenase as described above.

Further, the method further comprises: deactivating or deleting an original D-lactate dehydrogenase gene in the original strain; and blocking a pathway for synthesizing 2,3-butanediol by pyruvic acid.

Further, the method further comprises: reconstructing a promoter of the introduced nucleotide sequence into a constitutive promoter.

Further, the original strain is a ΔhsdR1ΔhsdR2 double-mutant strain MW3 of Bacillus licheniformis ATCC 14580; and α-acetolactate decarboxylase (alsD) and acetolactate synthase (alsS) genes are replaced with the nucleotide sequence as described above.

In still another aspect, the present invention further provides use of the above gene engineering strain, in particular use in production of D-lactic acid.

In one specific implementation mode, a carbon source adopted in the production is one or more selected from a group consisting of glucose, xylose, maltose, lactose and sucrose.

Preferably, low-price culture mediums containing peanut meal or dried corn steep liquor powder are adopted for fermentation in the production.

Preferably, fermentation temperature for the production is 45° C.-55° C.

Preferably, pH of the fermentation culture mediums in the production is 6.0-8.0.

Preferably, a fermentation process in the production is a continuous fermentation or fed-batch process.

In another aspect, the present invention provides a D-lactate dehydrogenase. In one specific implementation mode, the D-lactate dehydrogenase has one of the following amino acid sequences:

1) an amino acid sequence expressed by SEQ ID No. 1;

2) an amino acid sequence of a derivative protein produced from the amino acid sequence expressed by SEQ ID No. 1 through substitution, deletion or insertion of one or more amino acid residues, the derivative protein having D-lactate dehydrogenase activity; and

3) an amino acid sequence having at least about 80% of homology with the amino acid sequence expressed by SEQ ID No. 1.

The protein comes from T. indicus. The above protein may be artificially synthesized and may also be obtained by firstly synthesizing a coding gene thereof and then performing biological expression.

The present invention further provides nucleic acid molecules coding the above D-lactate dehydrogenase, and these nucleic acid molecules may be DNA such as cDNA, genome DNA or recombinant DNA; and may also be RNA such as mRNA, hnRNA or tRNA.

Further, the present invention provides a nucleotide sequence coding the above D-lactate dehydrogenase. The nucleotide sequence is one of the following nucleotide sequences:

1) a nucleotide sequence expressed by SEQ ID No. 2;

2) a nucleotide sequence expressed by SEQ ID No. 3; and

3) a nucleotide sequence having more than 80% of homology with the nucleotide sequence expressed by SEQ ID No. 2 or SEQ ID No. 3.

In another aspect, the present invention provides use of the above D-lactate dehydrogenase or the above nucleotide sequence in production of D-lactic acid.

In another aspect, the present invention provides a gene engineering strain, characterized in that the gene engineering strain has the D-lactate dehydrogenase according to claim 1 or a D-lactate dehydrogenase coded by the nucleotide sequence according to claim 2.

Further, in the above gene engineering strain, the L-lactate dehydrogenase gene is deactivated or deleted, and acetolactate synthase and/or acetolactate decarboxylase genes are deactivated or deleted.

Further, the above gene engineering strain further has one or more genes which are deactivated or deleted, and the one or more genes are selected from pyruvate formate-lyase gene, pyruvate formate-lyase activating enzyme gene, pyruvate dehydrogenase gene, acetokinase and ethanol dehydrogenase gene.

Further, an original strain of the above gene engineering strain is bacillus. Further, the bacillus are selected from a group consisting of Bacillus licheniformis, Bacillus coagulans, B. subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus circulans and Aneurinibacillus aneurinilyticus. Preferably, the original strain is Bacillus licheniformis. Preferably, the original strain is Bacillus licheniformis ATCC 14580. Preferably, the original strain is a ΔhsdR1ΔhsdR2 double-mutant strain MW3 of Bacillus licheniformis ATCC 14580.

Further, a promoter of the above D-lactate dehydrogenase gene is a promoter P_(als) of α-acetolactate synthase of Bacillus licheniformis ATCC 14580, a P_(c) promoter in plasmid pMMPc, a P₄₃ promoter in B. subtilis or a promoter P_(ldh) of L-lactate dehydrogenase of Bacillus licheniformis ATCC 14580. Preferably, the promoter is directly connected with an original codon “ATG” of ldh_(Ti) gene.

Further, the above gene engineering strain is Bacillus licheniformis BN11, the preservation number of which is CCTCC NO: M2016026 and which was preserved in China Center for Type Culture Collection on Jan. 8, 2016.

In still another aspect, the present invention provides a method for preparing the above gene engineering strain, comprising the following steps:

1) knocking out an L-lactate dehydrogenase gene in an original strain;

2) introducing the above nucleotide sequence coding the D-lactate dehydrogenase;

3) deactivating or deleting an acetolactate synthase gene and/or acetolactate decarboxylase gene; and

4) while or after performing step 3), adding a promoter sequence in the front of the nucleotide sequence introduced in step 2).

Specifically, by using a mutant strain MW3 of Bacillus licheniformis ATCC 14580 with the L-lactate dehydrogenase gene which is knocked out as a host, and replacing α-acetolactate decarboxylase (alsD) and acetolactate synthase (alsS) with the above D-lactate dehydrogenase, a heat-resistant engineering bacillus strain CCTCC NO: M2016026 capable of producing D-lactic acid through fermentation at temperature of 30° C.-55° C. by using glucose is obtained.

In yet another aspect, the present invention further provides use of the above gene engineering strain, in particular use in production of D-lactic acid. Further, the strain is used for producing D-lactic acid by using glucose, xylose, maltose, lactose, sucrose or a combination thereof as a carbon source. Further, low-cost culture mediums containing peanut meal and dried corn steep liquor powder are used for fermentation. Further, D-lactic acid is produced through fermentation under an environment condition between an anaerobic environment condition and a microaerobic environment condition with pH of 6.0-8.0 and temperature of 45° C.-55° C. Further, the fermentation process is a continuous fermentation or fed-batch process.

Further, firstly seed culture is performed to the above gene engineering strain to obtain seed culture solution, and then fermentation culture is performed in fermentation culture mediums using one of glucose, xylose, maltose, lactose and sucrose as a carbon source and using yeast powder, peptone, peanut meal and dried corn steep liquor as a nitrogen source to obtain D-lactic acid. Specifically, the process comprises the following steps:

1) slant culture: inoculating an engineered bacillus strain into a solid slant culture medium containing 20 g/L agar and culturing for 24-48 h at 45-55° C.;

2) seed culture: inoculating the engineered bacillus after slant culture into a seed culture medium under an aseptic condition, statically culturing for 24-36 h at 45-55° C. to obtain seed culture solution, wherein a neutralizing agent is added to control pH of fermentation liquid; and

3) fermentation culture: inoculating the seed culture solution into a fermentation culture medium in an inoculation amount according to a volume ratio of 5-20%, and culturing for 48-90 h at 45° C.-55° C., wherein the temperature is preferably 50° C.

Preferably, each liter of the seed culture medium in step 2) contains 60-120 g of glucose or 40-70 g of xylose, 8-12 g of yeast powder, 3-8 g of peptone, 50 g of calcium carbonate and balance of water, and preferably contains 90 g of glucose, 10 g of yeast powder, 5 g of peptone, 50 g of calcium carbonate and balance of water; and pH of the seed culture medium is 6.0. Sterilization is performed for 15 min at 115° C. The neutralizing agent comprises one or more of NaOH, NH₄OH and Ca(OH)₂.

Preferably, the fermentation culture medium in step 3) comprises the following components and contents thereof: 40-180 g/L carbon source and 5-20 g/L nitrogen source. Preferably, the fermentation process in step 3) is a fed-batch process, and the fed-batch process refers to that, when the total content of reducing sugars in the fermentation liquid is lower than 20 g/L, the carbon source is added to enable the total content of the reducing sugars to be maintained at 30-70 g/L or reach 50-70 g/L. Preferably, pH of the fermentation culture medium is 6.0-8.0.

The reconstructed gene engineering strain provided by the present invention can produce, by fermentation, high-concentration and highly optically-pure D-lactic acid at higher fermentation temperature by using low-cost raw materials, which can improve the production efficiency while the cost is reduced, so that the production cost is reduced and the method is suitable for application and dissemination in industrial production. The highest yield of D-lactic acid can reach 226 g/L with an optical purity of 99.9%, the highest sugar-acid conversion rate can reach 93.6% and the fermentation production capacity is 3.2 g/[L·h]. Therefore, by using the inventive method to produce D-lactic acid, the cost can be reduced, the operation flow can be simplified and the method has a wide industrial application prospect.

The concept, specific steps and produced technical effects of the present invention will be further described below with reference to the drawings, so as to fully understand the purposes, features and effects of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of construction of heat-resistant Bacillus licheniformis and a pathway to produce D-lactic acid according to one specific implementation mode of the present invention.

FIG. 2 is molecular weight of purified D-lactate dehydrogenase protein detected through SDS-PAGE, wherein lane M is a protein molecular weight standard; lane 1 shows crude enzyme liquid obtained after inducing E. coli BL21 (DE3) containing pETDuet-1 empty vectors; lane 2 shows crude enzyme liquid obtained after inducing E. coli BL21 (DE3) containing prokaryotic expression vectors pETDuet-ldh_(Ti); and lane 3 shows purified D-lactate dehydrogenase.

FIG. 3 is a determination result of the optimal reaction pH value s of D-lactate dehydrogenase.

FIG. 4 is a determination result of the optimal reaction temperature of D-lactate dehydrogenase.

FIG. 5 is construction of various plasmids: A. pKVMΔldh; B. pKVMN1; C. pKVMN2; D. pKVMN4; E. pKVMN6; and E pKVMA1.

FIG. 6 is optical purity analysis by HPLC for D-lactic acid produced by strains BN11.

FIG. 7 shows situations that strains BN11 use glucose with different concentrations to produce D-lactic acid through fermentation: A. situations for consumption of sugar with different concentrations; and B. situations for D-lactic acid produced by using sugar with different concentrations. Initial sugar concentration:

, 60.0 g/L; ▪, 87.0 g/L; ●, 122.0 g/L; ▴, 148.0 g/L; ▾, 180.0 g/L; and ♦, 202.0 g/L.

FIG. 8 is shows situations that strains BN11 produce D-lactic acid through batch fermentation and fed (glucose)-batch fermentation: A. batch fermentation results; and B. fed (glucose)-batch fermentation results. ▪, cell density; ●, glucose; ▴, D-lactic acid; ♦, formic acid; ▾, acetic acid; and ★, ethanol.

FIG. 9 is shows situations that strains BAH produce D-lactic acid through batch fermentation and fed (glucose)-batch fermentation: A. batch fermentation results; and B. fed (glucose)-batch fermentation results. ▪, cell density; ●, glucose; ▴, D-lactic acid; ♦, formic acid; ▾, acetic acid; and ★, ethanol.

FIG. 10 is shows situations that strains BN11 produce D-lactic acid through fed-batch fermentation by using xylose. ▪, cell density; ●, xylose; ▴, D-lactic acid; ♦, formic acid; ▾, acetic acid; and ★, ethanol.

FIG. 11 shows situations that strains BN11 produce D-lactic acid through fed (glucose)-batch fermentation by using low-cost culture mediums. ▪, cell density; ●, glucose; ▴, D-lactic acid; ♦, formic acid; ▾, acetic acid; and ★, ethanol.

FIG. 12 is a phylogenetic tree of D-lactate dehydrogenases predicted in partial typical thermophilus.

FIG. 13 shows analysis results of enzyme catalytic sites obtained through amino acid sequence comparison between an amino acid sequence (query) expressed by SEQ ID No. 1 and other D-lactate dehydrogenases (SEQ ID Nos: 85-93).

FIG. 14 shows analysis results of ligand binding sites obtained through amino acid sequence comparison between an amino acid sequence (query) expressed by SEQ ID No. 1 and other D-lactate dehydrogenases (SEQ ID Nos: 85-93).

FIG. 15 shows analysis results of NAD binding sites obtained through amino acid sequence comparison between an amino acid sequence (query) expressed by SEQ ID No. 1 and other D-lactate dehydrogenases (SEQ ID Nos: 85-93).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In one aspect, the present invention provides a D-lactate dehydrogenase, which is capable of catalyzing pyruvic acid to synthesize D-lactic acid.

In one preferred specific implementation mode, the D-lactate dehydrogenase has one of the following amino acid sequences:

1) an amino acid sequence expressed by SEQ ID No. 1;

2) an amino acid sequence derivatively produced from the amino acid sequence expressed by SEQ ID No. 1 through substitution, deletion, insertion or addition of one or more amino acid residues;

3) an amino acid sequence produced from the amino acid sequence expressed by SEQ ID No. 1 through conservative replacement; and

4) an amino acid sequence having at least 80% of homology with the amino acid sequence expressed by SEQ ID No. 1.

“Conservative replacement” described here should be understood as those substitutions in which given amino acids in a polypeptide are substituted with another type of amino acids with similar features. Typically, the following replacement is considered as conservative substitution: replacing aliphatic amino acids such as Ala, Val, Leu and Ile with another aliphatic amino acids; replacing Ser with Thr, vice versa; replacing acidic residues of Asp or Glu with another acidic residues; replacing acylamino-containing residues of Asn or Gln with another acylamino-containing residues; replacing alkaline residues of Lys or Arg with another alkaline residues; and replacing aromatic residues of Phe or Tyr with another aromatic residues.

Functionally equivalent amino acids generally are similar to amino acids replaced thereby in aspects of size and/or feature (such as charge or hydrophobicity) Amino acids with similar features can be grouped as follows:

(1) hydrophobic amino acids: His, Trp, Trp, Tyr, Phe, Met, Leu, Ile, Val, and Ala;

(2) neutrally hydrophobic amino acids: Cys, Ser, and Thr;

(3) polar amino acids: Ser, Thr, Asn, and Gln;

(4) acidic/negatively charged amino acids: Asn, Lys, and His;

(5) charged amino acids: Asp, Glu, Asn, Lys, and His;

(6) alkaline/positively charged amino acids: Asn, Lys, and His;

(7) alkaline amino acids: Asn, Gln, His, Lys, and Arg;

(8) amino acids with residues influencing chain orientations: Gly, Pro; and

(9) aromatic amino acids: Trp, Tyr, Phe, and His.

A phylogenetic tree, drawn by using MEGA 5 software and a neighbor-joining method, of amino acid sequences of D-lactate dehydrogenases predicted in partial typical thermophilus is as illustrated in FIG. 12, wherein a strain expressed with a bold font is a donor strain of the D-lactate dehydrogenase expressed by SEQ ID No. 1, the D-lactate dehydrogenase having the lowest homology with the D-lactate dehydrogenase expressed by SEQ ID No. 1 is a D-lactate dehydrogenase in Truepera radiovictrix, and the similarity between the two amino acids for the two D-lactate dehydrogenases is 28%; and the D-lactate dehydrogenase having the highest homology with the D-lactate dehydrogenase expressed by SEQ ID No. 1 is a D-lactate dehydrogenase in Treponema caldarium DSM 7334, and the similarity between the two amino acids for the two D-lactate dehydrogenase is 45%.

Preferably, the substitution, deletion, insertion or addition of the amino acid residues occurs outside a functional domain comprising enzyme catalytic sites, ligand binding sites and NAD binding sites of the D-lactate dehydrogenase Amino acid sequence comparison is performed between the amino acid sequence expressed by SEQ ID No. 1 and other D-lactate dehydrogenases, so as to obtain comparison analysis results for catalytic sites, ligand binding sites and NAD binding sites as illustrated in FIG. 13, FIG. 14 and FIG. 15.

More preferably, the substitution, deletion, insertion or addition of the amino acid residues occurs outside key amino acids at enzyme catalytic sites, ligand binding sites and NAD binding sites. Herein, key amino acids at the enzyme catalytic sites are Arg at position 229, Glu at position 258 and His at position 290; key amino acids at the ligand binding sites are Ser, Ala and Gly positions 75-77, Tyr at position 99, Arg at position 229, His at position 290 and Phe at position 293; and key amino acids at the NAD binding sites are Tyr at position 99, Ile at position 104, Gly at position 150, Gly, Lys, Ile and Gly at positions 152-155, Tyr, Asp and Pro at positions 172-174, His, Cys, Pro and Leu at positions 199-202, Asn at position 206, Met at position 209, Thr, Ala and Arg at positions 227-229, Asp and Val at positions 253 and 254, His at position 290, and Ala and Phe at positions 292 and 293.

In another aspect, the present invention provides a nucleotide sequence coding the above D-lactate dehydrogenase.

In one preferred specific implementation mode, the nucleotide sequence is one of the following nucleotide sequences:

1) a nucleotide sequence expressed by SEQ ID No. 2;

2) a nucleotide sequence expressed by SEQ ID No. 3;

3) a nucleotide sequence having more than 80% of homology with the nucleotide sequence expressed by SEQ ID No. 2 or SEQ ID No. 3; and

4) a nucleotide sequence hybridized with a complementary chain of the nucleotide sequence expressed by SEQ ID No. 2 or SEQ ID No. 3 under a high-stringency condition.

“Hybridization under low-stringency, medium-stringency, high-stringency or extremely-high-stringency condition” described herein describes conditions for hybridization and washing. Guidance for performing a hybridization reaction can be seen in Current Protocols in Molecular Biology, John Wiley&Sons, N.Y. (1989), 6.3.1-6.3.6. In this literature, a liquid-phase hybridization method and a non-liquid-phase hybridization method are described and either of them is usable. Specific hybridization conditions herein are as follows: 1) a low-stringency hybridization condition refers to hybridization at about 45° C. in 6× sodium chloride/sodium citrate (SSC), and then washing twice at 50° C. at least (for low-stringency conditions, washing temperature shall be increased to 55° C.) in 0.2×SSC, and 0.1% SDS; 2) a medium-stringency hybridization condition refers to hybridization at about 45° C. in 6×SSC, and then washing one or more times at 60° C. at least in 0.2×SSC, and 0.1% SDS; 3) a high-stringency hybridization condition refers to hybridization at about 45° C. in 6×SSC, and then washing one or more times at 65° C. in 0.2×SSC, and 0.1% SDS; and 4) an extremely-high-stringency hybridization condition refers to hybridization at 65° C. in 0.5M sodium phosphate, and 7% SDS, and then washing one or more times at 65° C. at least in 0.2×SSC, and 0.1% SDS. The high-stringency condition (3) is preferred and should be used unless otherwise specially pointed out.

In another aspect, the present invention provides a gene engineering strain, which has the D-lactate dehydrogenase as described above or a D-lactate dehydrogenase coded by the nucleotide sequence as described above.

In one specific implementation mode, in the original strain of the gene engineering strain, a pathway for synthesizing L-lactic acid by pyruvic acid and a pathway for synthesizing 2,3-butanediol by pyruvic acid are blocked. In one embodiment, the pathway for synthesizing L-lactic acid by pyruvic acid is blocked by deactivating or deleting an L-lactate dehydrogenase gene; and the pathway for synthesizing 2,3-butanediol by pyruvic acid is blocked by deactivating or deleting one or both of an acetolactate synthase gene and an acetolactate decarboxylase gene.

In another specific implementation mode, in addition to the blocked pathways as mentioned above, one or more of a pathway for synthesizing formic acid by pyruvic acid, a pathway for synthesizing acetic acid by pyruvic acid and a pathway for synthesizing ethanol by pyruvic acid are blocked in the original strain. The pathway for synthesizing formic acid by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate formate-lyase gene and a pyruvate formate-lyase activating enzyme gene; the pathway for synthesizing acetic acid by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate dehydrogenase gene and an acetokinase gene; and the pathway for synthesizing ethanol by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate dehydrogenase gene and an ethanol dehydrogenase gene.

Preferably, an original strain is thermophilus. More preferably, a restrictive modification system in the original strain is deactivated or knocked out.

Preferably, a promoter of a gene of the above D-lactate dehydrogenase is reconstructed into a constitutive promoter, i.e., no inducer is needed; and the regulation of the constitutive promoter is not influenced by external conditions and the expression of the promoter gene has continuity.

FIG. 1 is a schematic diagram of construction of D-lactic acid engineered strain and a pathway to produce D-lactic acid according to one embodiment.

“Block” herein refers to blocking a certain pathway through various gene engineering methods, including deactivating or deleting genes for one or more catalytic enzymes in this pathway such that this pathway is unworkable.

“Original D-lactate dehydrogenase gene” herein refers to a D-lactate dehydrogenase gene carried by the original strain itself.

One skilled in the art can understand that the original strain may also not contain the deleted or deactivated enzyme gene or the blocked pathways, such as L-lactate dehydrogenase gene and D-lactate dehydrogenase gene or the like. Thus, when the gene engineering strain is constructed, these genes or pathways are naturally deleted, and are not required to be deactivated, deleted or blocked by performing additional gene engineering operations.

In another aspect, the present invention provides use of the above gene engineering strain, in particular use in production of D-lactic acid.

In one specific implementation mode, the gene engineering strain may produce D-lactic acid through fermentation by using a carbon source which is one or more selected from a group consisting of glucose, xylose, maltose, lactose and sucrose; and may also produce D-lactic acid through fermentation by using low-cost culture mediums containing peanut meal and dried corn steep liquor powder. Since the original strain for the gene engineering strain is thermophilus and the original source of the introduced D-lactate dehydrogenase is also thermophilus, the gene engineering strain can produce D-lactic acid at higher fermentation temperature.

The present invention will be further described in detail below in combination with the embodiments.

Unless otherwise specially stated, experimental methods used in the following embodiments are all conventional methods. Unless otherwise specially stated, materials, reagents, strains and the like used in the following embodiments can be commercially available.

Reagents and strains: all reagents in the present invention were reagents which were commercially available and reagent grade or higher. Herein, FastPfu DNA polymerases were available from Beijing TransGen Biotech Co., Ltd. All restriction endonucleases and T4 DNA ligases were available from NEB (New England Biolabs). Isopropyl-beta-D-thiogalactopyranoside (IPTG), dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) were available from Merck KGaA. L-lactic acid and D-lactic acid standard products were available from Sigma-Aldrich Corporation. pMD18-T and all primers were synthesized in TaKaRa (Dalian). Bacillus licheniformis ATCC 14580 may be directly available from ATCC website, double-mutant strains MW3 (ΔhsdR1, ΔhsdR2) of Bacillus licheniformis ATCC 14580 may be constructed according to the method in literature Generation of Readily Transformable Bacillus Licheniformis Mutants (Bianca Waschkaw et al., Appl Microbiol Biotechnol, (2008) 78:181-188), and the double-mutant strains MW3 were used as host bacteria during DNA operations. E. coli DH5a and BL21 (DE3) were used as cloning host and expression host bacterium, respectively. E. coli S17-1 were used as donor bacteria for conjugal transfer. pETDuet-1 was used as an expression vector. Shuttle plasmid pKVM1 was resistant to penbritin and erythrocin and used for knocking out genes of Bacillus licheniformis MW3. Luria-Bertani (LB) culture mediums were used for culturing E. coli and bacillus. Penbritin (100 g/mL), erythrocin (5 g/mL) and polymyxin B (40 g/mL) were used for screening E. coli and bacillus. X-Gal (40 g/mL) was used for blue-white screening.

HPLC analysis for D-lactic acid produced by strains was performed by using a chiral column MCI GEL CRS10W.

Embodiment 1: Obtaining of D-Lactate Dehydrogenase Gene and Protein Thereof

1. Obtaining of D-lactate Dehydrogenase Ldh_(Ti) Gene

By using D-lactate dehydrogenase NP_213499 in Aquifex aeolicus VF5 as a template in NCBI, a protein WP_013906894 which had a similarity of 38% with NP_213499 and was possibly D-lactate dehydrogenase was obtained through comparison, and a sequence thereof was as expressed by SEQ ID No. 1. Through further search, it was found that this protein existed in Thermodesulfatator indicus DSM 15286, a corresponding nucleotide sequence thereof was a complete open reading frame, as expressed by SEQ ID No. 2, with a length of 978 bp, coding a protein consisting of amino acid residues expressed by SEQ ID No. 1, and the protein was named as Ldh_(Ti). After optimization according to the codon of E. coli K12, a synthesis process was performed by a PCR synthesis method, and by adopting primers shown in Table 1 in which the primers served as templates for each other, thereby synthesizing a nucleotide sequence optimized by the codon with a sequence thereof as expressed by SEQ ID No. 3. The obtained gene as expressed by SEQ ID No. 3 was inserted into a pMD18-T vector to obtain a pMD18-T-ldh_(Ti) plasmid, and nucleotide sequence determination was performed. One skilled in the art can understand that the gene expressed by SEQ ID No. 3 may also be directly synthesized by means of gene synthesis.

TABLE 1 Sequences of primers for synthesis of D-lactate dehydrogenase Ldh_(Ti) through PCR Pri- mer Sequence (5′-3′)  P1 CCGCGGATCCGATGAAAGTAATTTTTTT (SEQ ID No. 4) P2 TCTTCATACGGGTGCATAGAAAAAAAAAT TACTTTCATCGGATCCG (SEQ ID No. 5) P3 TTCTATGCACCCGTATGAAGAGGAATTTC TGGGTCCGATTCTGCC (SEQ ID No. 6) P4 GGGGTCATTTCTACGTCCCAGTCAGACGG CAGAATCGGACCCAGA (SEQ ID No. 7) P5 GGGACGTAGAAATGACCCCGGACTTTCTG GACGAAACCACCGTGG (SEQ ID No. 8) P6 CTTACTACCTGGGCACCTTTAGCCTTTTC CACGGTGGTTTCGTCC (SEQ ID No. 9) P7 TAAAGGTGCCCAGGTAGTAAGCCTGTTTG TTTCTGACAAAGCTGA (SEQ ID No. 10) P8 GCAGCGCTTCCAGTACCGGACCATCAGCT TTGTCAGAAACAAACA (SEQ ID No. 11) P9 GGTACTGGAAGCGCTGCATTCTTACGGTG TGGGCCTGCTGGCCCT (SEQ ID No. 12) P10 AATATCGATGTGATCATAGCCAGCAGAA CGCAGGGCCAGCAGGCC (SEQ ID No. 13) P11 CTGGCTATGATCACATCGATATTGAGAC CGCAAAACGCCTGGGTA (SEQ ID No. 14) P12 GAATAGGCTGGCACGTTAACTACTTTGA TACCCAGGCGTTTTGCG (SEQ ID No. 15) P13 AGTTAACGTGCCAGCCTATTCTCCGCAC GCTATCGCTGACCATAC (SEQ ID No. 16) P14 AATCAGAGCCAGCATGATAGCCAGAGTA TGGTCAGCGATAGCGTG (SEQ ID No. 17) P15 GCTATCATGCTGGCTCTGATTCGTCGTC TGCACCGTGCCCATGAT (SEQ ID No. 18) P16 CCAGATCAAAATCACCCAGGCGCACTTT ATCATGGGCACGGTGCA (SEQ ID No. 19) P17 CCTGGGTGATTTTGATCTGGATGGTCTG ATGGGCTTTGATCTGAA (SEQ ID No. 20) P18 CCAATTACACCAGCAACTTTGCCGTTCA GATCAAAGCCCATCAGA (SEQ ID No. 21) P19 GCAAAGTTGCTGGTGTAATTGGTCTGGG TAAAATCGGTCGCCTGG (SEQ ID No. 22) P20 ACCAAACGCTTTCAGGCGGGTAGCTACC AGGCGACCGATTTTACC (SEQ ID No. 23) P21 CGCCTGAAAGCGTTTGGTTGCAAAGTTC TGGGCTATGATCCATAC (SEQ ID No. 24) P22 TTTTCTACGATTTCCGGCTGAATGTATG GATCATAGCCCAGAACT (SEQ ID No. 25) P23 TCAGCCGGAAATCGTAGAAAACGTTGAT CTGGATACCCTGATCAC (SEQ ID No. 26) P24 ATGAATAGAAATGATATCAGCCTGAGTG ATCAGGGTATCCAGATCAA (SEQ ID No. 27) P25 TCAGGCTGATATCATTTCTATTCATTGT CCGCTGACCCGTGAAAA (SEQ ID No. 28) P26 AAAGTCTCTTCGTTAAACATATGAAAGT TTTCACGGGTCAGCGGA (SEQ ID No. 29) P27 CTTTCATATGTTTAACGAAGAGACTTTT AAGCGTATGAAACCGGGTG (SEQ ID No. 30) P28 CCACGCGCGGTGTTAACCAGAATAGCAC CCGGTTTCATACGCTTA (SEQ ID No. 31) P29 GTTAACACCGCGCGTGGTGGTCTGATCG ATACCAAGGCCCTGCTG (SEQ ID No. 32) P30 GCCCAGTTTACCAGACTTCAGGGCCTCC AGCAGGGCCTTGGTATC (SEQ ID No. 33) P31 CTGAAGTCTGGTAAACTGGGCGGCGCAG CCCTGGATGTGTATGAA (SEQ ID No. 34) P32 TTTTAAAAAACAGGCCACGTTCATATTC ATACACATCCAGGGCTG (SEQ ID No. 35) P33 GAACGTGGCCTGTTTTTTAAAAACCACC AAAAAGAAGGTATCAAA (SEQ ID No. 36) P34 GCAGCTGGGCCAGATACGGGTCTTTGAT ACCTTCTTTTTGGTGGT (SEQ ID No. 37) P35 CGTATCTGGCCCAGCTGCTGGGTCTGGC CAACGTAGTGCTGACCG (SEQ ID No. 38) P36 CCTCACGGGTCAGAAAGGCCTGATGACC GGTCAGCACTACGTTGG (SEQ ID No. 39) P37 CCTTTCTGACCCGTGAGGCTGTAAAAAA CATCGAAGAAACTACCG (SEQ ID No. 40) P38 TTGCCATTCCAGAATGTTTTCTACGGTA GTTTCTTCGATGTTTTTTAC (SEQ ID No. 41) P39 TAGAAAACATTCTGGAATGGCAAAAGAA CCCGCAGGCAAAGCTGA (SEQ ID No. 42) P40 GCCCAAGCTTTTAGATTTCGTTTTTCAG CTTTGCCTGCGG (SEQ ID No. 43) Underlines represents enzyme cutting sites.

2. Construction of Recombinant Prokaryotic Expression Vector

PCR amplification was performed to the obtained pMD18-T-ldh_(Ti) plasmid by using primers 1 and 40 as shown in Table 1, the obtained target gene segments were digested with two enzymes BamHI and HindIII, then the segments were linked to an expression vector pETDuet-1 (Novagen) which was also subjected to a double enzyme digestion treatment using the same enzymes, a linked product containing the Ldh_(Ti) gene was transformed into E. coli BL21 (DE3) (Novagen), and after a PCR process and an identification process by DNA sequencing, a plasmid in a positive clone containing correct Ldh_(Ti) gene was named as pETDuet-ldh_(Ti), and it can be used for expression of the D-lactate dehydrogenase.

3. Prokaryotic Expression and Purification of D-Lactate Dehydrogenase

1) Prokaryotic Expression of D-Lactate Dehydrogenase Ldh_(Ti)

The obtained E. coli BL21 (DE3) containing the prokaryotic expression vector pETDuet-ldh_(Ti) was shaken at 37° C. till OD_(600 nm) was 0.6-0.8, and then was added with IPTG with a final concentration of 1 mM for induction, the obtained mixture was in a shaker for culture for 16 h at 16° C. or for 8 h at 30° C., then the cultured bacteria cells were collected by centrifugation, resuspended in 50 mM phosphate buffer solution (PBS; pH 7.4), and broken by an ultrasonic wave manner, and then the supernatant was collected for SDS-PAGE electrophoretic analysis.

Results are as shown in FIG. 2, from which it can be seen that the prokaryotic expression vectors pETDuet-ldh_(Ti), carrying the target gene are abundantly expressed in E. coli BL21 (DE3), and the single subunit molecular weight of the expressed recombinant protein is about 37 kDa, which is compliant with the expected result.

2) Purification of D-Lactate Dehydrogenase Ldh_(Ti)

The proteins expressed in step 1) were purified through His-tag, which was specifically as follows:

The prokaryotic expression product obtained in step 1), which was detected by SDS-PAGE, was purified by affinity chromatography, i.e., the supernatant after ultrasonication was passed through a chromatographic column filled with Ni-NTA gel, the proteins containing His-tag were bound to the Ni-NTA gel, then non-specifically bound impure proteins were washed away by cleaning buffer solution (25 mM Tris hydrochloric acid buffer solution, 500 mM NaCl, and 50 mM imidazole, wherein the above-mentioned concentrations were final concentrations in solution, PH 8.0), and the target proteins were finally eluted by elution buffer solution (25 mM Tris hydrochloric acid buffer solution, 500 mM NaCl, and 220 mM imidazole, wherein the above-mentioned concentrations were final concentrations in solution, PH 8.0) to obtain the purified target proteins. Then, by using a molecular sieve gel column and replacing the buffer solution with 50 mM phosphate buffer solution (pH 7.0), the final purified target proteins D-lactate dehydrogenase Ldh_(Ti) was obtained.

SDS-PAGE electrophoresis was performed to the final purified target proteins D-lactate dehydrogenase Ldh_(Ti), and a result is as shown in lane 3 of FIG. 2, wherein the target band is single, and the size is about 37 kDa, indicating that the obtained target proteins are relatively pure.

Embodiment 2: Enzymatic Characteristic Identification of Target Protein D-Lactate Dehydrogenases Ldh_(ti) Obtained in Embodiment 1

The enzymatic characteristic of the target protein obtained in Embodiment 1 was identified by using the following substrates: pyruvic acid, D-lactic acid, L-lactic acid, glyceric acid, phenylpyruvic acid, glyoxylic acid and oxaloacetic acid. A reaction system is consisting of: 50 mM phosphate buffer solution (pH 7.0), 0.2 mM NADH or NAD⁺ as coenzyme, a suitable amount of D-lactate dehydrogenases Ldh_(Ti) and substrates with different concentrations, reacted at 37° C. An enzyme activity determination method is to determine oxidization of NADH or reduction of NAD at ultraviolet wavelength of 340 nm, wherein one enzyme activity unit is defined as the enzyme amount needed for oxidizing or reducing 1 μmoL NADH or NAD in one minute.

Results show that the target proteins obtained in Embodiment 1 can catalyze pyruvic acid, glyoxylic acid, oxaloacetic acid, D-lactic acid and phenylpyruvic acid, but have no activity for L-lactic acid and glyceric acid (Table 2). Particularly, D-lactate dehydrogenases Ldh_(Ti) have the highest catalytic efficiency for pyruvic acid and can specifically catalyze D-lactic acid. Therefore, the above-mentioned results show that the target proteins obtained in Embodiment 1 are D-lactate dehydrogenases Ldh_(Ti) indeed.

TABLE 2 Ranges for Substrate Spectrums Catalyzed by D-lactate Dehydrogenases Ldh_(Ti) K_(m) k_(cat) k_(cat)/K_(m) Substrate mM min⁻¹ M⁻¹ min⁻¹ Pyruvic Acid 0.05 ± 0.01 64.7 ± 1.70 (1.24 ± 0.04) × 10⁶ L-lactic Acid — — — D-lactic Acid 5.41 ± 0.28 6.29 ± 0.30 (1.16 ± 0.03) × 10³ Glyoxylic 1.76 ± 0.08 137.8 ± 3.10  (7.83 ± 0.29) × 10⁴ Acid Oxaloacetic 1.32 ± 0.05 268.4 ± 5.90  (2.03 ± 0.07) × 10⁵ Acid Phenylpyruvic 4.58 ± 0.24 17.7 ± 0.68 (3.86 ± 0.22) × 10³ Acid Glyceric Acid — — — “—” represents that the enzyme has no activity for the substrate.

Herein, the optimal reaction pH value and the optimal reaction temperature for the D-lactate dehydrogenases Ldh_(Ti) obtained in Embodiment 1 were determined through the following two tests:

1) Determination of the optimal Reaction pH Value of D-lactate Dehydrogenases Ldh_(Ti)

An enzyme activity determination method is to determine change in NADH at ultraviolet wavelength of 340 nm, and one enzyme activity unit is defined as the enzyme amount needed for oxidizing 1 μmoL of NADH in one minute. At 37° C., by using 20 mM pyruvic acid as a substrate and 0.2 mM NADH as a coenzyme, the change of enzyme activity of the D-lactate dehydrogenase within a pH range of 3.0-11.0 was determined. A buffer system is consisting of: 50 mM citric acid-sodium citrate buffer solution with pH of 3.0-7.0; 50 mM phosphate buffer solution with pH of 7.0-9.0; and 50 mM sodium carbonate-sodium hydrogen carbonate buffer solution with pH of 9.0-11.0. Experiments were repeated thrice. A result is as shown in FIG. 3, wherein the optimal reaction pH value of this enzyme is 6.0. Relative enzyme activities in FIG. 3 are relative enzyme activities, which are obtained by: determining enzyme activities at different pH values, and correspondingly converting the enzyme activity values at other pH values on the basis that the highest value at pH 6.0 is 100%.

2) Determination of the Optimal Reaction Temperature of D-lactate Dehydrogenases Ldh_(Ti)

An enzyme activity determination method is to determine change in NADH at ultraviolet wavelength of 340 nm, and one enzyme activity unit is defined as the enzyme amount needed for oxidizing 1 μmoL of NADH in one minute. Conditions for determination of the optimal reaction temperature of D-lactate dehydrogenases were as follows: 20 mM pyruvic acid as a substrate, 0.2 mM NADH as a coenzyme and 20 mM phosphate buffer solution (pH 7.0) system are adopted; and enzyme activities from 30° C. to 100° C. were respectively determined, and experiments were repeated thrice. A result is as shown in FIG. 4, wherein the optimal reaction temperature of the enzyme is 70° C. Relative enzyme activities in FIG. 4 are relative enzyme activities, which are obtained by: determining enzyme activities at different temperature conditions, and correspondingly converting the enzyme activity values at other temperature conditions on the basis that the highest value at 70° C. is 100%.

Embodiment 3: Preparation of Heat-resistant Bacillus Licheniformis Strain

1. Construction of Various Knockout Plasmids in the Present Invention

1) Construction of Knockout Plasmids for an L-lactate Dehydrogenase Gene: by using ATCC 14580 genome DNA as a template and primer pairs ldh-up-F/ldh-up-R and ldh-Dn-F/ldh-Dn-R, PCR amplification was performed respectively to upstream and downstream homologous arms of the L-lactate dehydrogenase gene, then the upstream and downstream homologous arms after amplification were respectively subjected to double enzyme digestion with BamHI/XhoI and XhoI/NcoI, simultaneously plasmid pKVM1 was subjected to double enzyme digestion with BamHI/NcoI, the above vector and segments after enzyme digestion were linked by using T4 DNA ligase and transformed into E. coli S17-1, and after correctness was verified through DNA sequencing, a positive clone plasmid was named as pKVMΔldh, with plasmid construction as shown in FIG. 5. Herein, the plasmidpKVM1 may be constructed according to the literature Size Unlimited Markerless Deletions by a Transconjugative Plasmid-system in Bacillus Licheniformis (Rachinger, M. et al., Journal of biotechnology, 2013, 167(4), 365-369).

2) Construction of Knockout Plasmids for Acetolactate Synthase (alsS) and Acetolactate Decarboxylase (alsD) Genes in Metabolic Pathways of 2,3-Butanediol

Herein, used primer sequences are as shown in Table 3.

(1) By using ATCC 14580 genome DNA as a template and primer pairs Als1-up-F/Als1-up-R and Als-Dn-F/Als1-Dn-R, PCR amplification was respectively performed to upstream and downstream homologous arms, simultaneously PCR amplification was performed to a D-lactate dehydrogenase Ldh_(Ti) gene by a primer pair Als-ldh-F/Als-ldh-R, and then the above-mentioned three gene segments were fused by primers Als1-up-F and Als1-Dn-R according to a recombinant PCR method. The above-mentioned recombinant PCR product and Als1-Dn-R were respectively subject to double enzyme digestion with enzymes BamHI/NcoI, the product linked by using T4 DNA ligase was transformed into E. coli S17-1, and after correctness was verified through DNA sequencing, a positive clone plasmid was named as pKVMN1 (FIG. 5B). An upstream homologous arm end of this plasmid completely reserved a promoter region (P_(als)) of alsS and was directly linked with an original codon “ATG” of ldf_(Ti). Herein, genome sequences of ATCC 14580 may be obtained from NCBI. Herein, a sequence of P_(als) is as expressed by SEQ ID No. 69.

(2) Construction of Knockout Plasmids Using P_(c) Promoter for Originating ldh_(Ti) Expression: by using plasmid pMMPc as a template and primers Als2-Pc-F and Als2-Pc-R, PCR amplification was performed to obtain a gene of P_(c) promoter. Herein, the plasmid pMMPc may be constructed from the plasmid pMMB66EH according to the method in the literature New Constitutive Vectors: Useful Genetic Engineering Tools for Biocatalysis (Xu, Y., Tao, F., Ma, C., & Xu, P., Applied And Environmental Microbiology, 2013, 79(8), 2836-2840); and by using ATCC 14580 genome DNA as a template and primer pairs Als2-up-F/Als2-up-R and Als-Dn-F/Als2-Dn-R, PCR amplification was respectively performed to upstream and downstream homologous arms, simultaneously PCR amplification was performed to a D-lactate dehydrogenase Ldh_(Ti) gene by using a primer pair Als2-ldh-F/Als-ldh-R, and then the above-mentioned four gene segments were fused with primers Als2-up-F and Als2-Dn-R according to a recombinant PCR method. The above-mentioned recombinant PCR product and pKVM1 were respectively subjected to double enzyme digestion with enzymes BamHI/XmaI, the product linked by using T4 DNA ligase was transformed into E. coli S17-1, and after correctness was verified through DNA sequencing, a positive clone plasmid was named as pKVMN2 (FIG. 5C). Herein, a sequence of P_(c) is as expressed by SEQ ID No. 70.

(3) Construction of Knockout Plasmids Using P₄₃ Promoter for Originating ldh_(Ti) Expression: by using B. subtilis genome DNA as a template and primers Als4-P₄₃-F and Als4-P₄₃-R, PCR amplification was performed to obtain a gene for the P₄₃ promoter, wherein information about P₄₃ promoter may be known from the literature Isobutanol Production at Elevated Temperatures in Thermophilic Geobacillus Thermoglucosidasius (Lin, P. P., Rabe, K. S., Takasumi, J. L., Kadisch, M., Arnold, F. H., & Liao, J. C., Metabolic Engineering, 2014, 24, 1-8); and by using ATCC 14580 genome DNA as a template and primer pairs Als2-up-F/Als4-up-R and Als-Dn-F/Als2-Dn-R, PCR amplification was respectively performed to upstream and downstream homologous arms, simultaneously PCR amplification was performed to a D-lactate dehydrogenase Ldh_(Ti) gene by using a primer pair Als2-ldh-F/Als-ldh-R, and then the above-mentioned four gene segments were fused by using primers Als2-up-F and Als2-Dn-R according to a recombinant PCR method. The above-mentioned recombinant PCR product and pKVM1 were respectively subjected to double enzyme digestion with enzymes BamHI/XmaI, the product linked by using T4 DNA ligase was transformed into E. coli S17-1, and after correctness was verified through DNA sequencing, a positive clone plasmid was named as pKVMN4 (FIG. 5D). Herein, a sequence of P₄₃ is as expressed by SEQ ID No. 71.

(4) Construction of Knockout Plasmids Expressed by Starting ldh_(Ti) with P_(ldh) Promoter: P_(ldh) was a promoter of Bacillus licheniformis ATCC 14580 L-lactate dehydrogenase. By using ATCC 14580 genome DNA as a template and primers Als6-Pldh-F and Als6-Pldh-R, PCR amplification was perform to obtain a gene of the P_(ldh) promoter; and by using primer pairs Als2-up-F/Als6-up-R and Als-Dn-F/Als2-Dn-R, PCR amplification was respectively performed to upstream and downstream homologous arms, simultaneously PCR amplification was performed to a D-lactate dehydrogenase Ldh_(Ti) gene by using a primer pair Als2-ldh-F/Als-ldh-R, and then the above-mentioned four gene segments were fused by using primers Als2-up-F and Als2-Dn-R according to a recombinant PCR method. The above-mentioned recombinant PCR product and pKVM1 was respectively subjected to double enzyme digestion with enzymes BamHI/XmaI, the product linked by using T4 DNA ligase was transformed into E. coli S17-1, and after correctness was verified through DNA sequencing, a positive clone plasmid was named as pKVMN6 (FIG. 5E). Herein, a sequence of P_(ldh) is as expressed by SEQ ID No. 72.

(5) Construction of Knockout Plasmids with Replacing ldh_(Ti) with Mesophilic D-lactate Dehydrogenase Gene: by using ATCC 14580 genome DNA as a template and primer pairs Als1-up-F/AlsA-up-R and Als-Dn-F/Als1-Dn-R, PCR amplification was respectively performed to upstream and downstream homologous arms; and by using E. coli K12 genome DNA as a template and primers AlsA-ldhA-F/AlsA-ldhA-R, PCR amplification was performed to a D-lactate dehydrogenase LdhA gene, and then by using primers Als1-up-F and Als1-Dn-R, the above-mentioned three gene segments were fused according to a recombinant PCR method. The above-mentioned recombinant PCR product and pKVM1 were respectively subjected to double enzyme digestion with enzymes BamHI/NcoI, the product linked by using T4 DNA ligase was transformed into E. coli S17-1, and after correctness was verified through DNA sequencing, a positive clone plasmid was named as pKVMA1 (FIG. 5F).

TABLE 3 Primers used for vector construction Name Sequence (5′-3′) ldh- TACGGGATCCTCGGCGACCGATGAACCGAACT up-F (SEQ ID NO: 44) ldh- TCCGCTCGAGGACTCATCATTCCTTTGCCG up-R (SEQ ID NO: 45) ldh- CCGCTCGAGGGCGTAACTGAACACCATGA Dn-F (SEQ ID NO: 46) ldh- CATGCCATGGCAAAGAAAGCGATGACCGGCA Dn-R (SEQ ID NO: 47) Als1- ACGCGGATCCCTTTGGCAATGACGATCAGCGA up-F (SEQ ID NO: 48) Als1- TGCATAGAAAAAAAAATTACTTTCATAGCCCT up-R CACTCCTCCATTTTCA (SEQ ID NO: 49) Als- ATGAAAAAGCCCTCTTTGAAAAGGGGGC Dn-F (SEQ ID NO: 50) Als1- CATGCCATGGGGTTTCATAAGACCGCTGATGA Dn-R (SEQ ID NO: 51) Als1- ATGAAAGTAATTTTTTTTTC ldh-F (SEQ ID NO: 52) Als- CCCCTTTTCAAAGAGGGCTTTTTCATTTAGAT ldh-R TTCGTTTTTCAGCT (SEQ ID NO: 53) Als2- ACGCGGATCCGCAAACAGCTGTTCATGAACCG up-F (SEQ ID NO: 54) Als2- AACGCGCTGTTGTTCTTGTATCGGCACGGGTA up-R CATTTGAAGGATCTTG (SEQ ID NO: 55) Als2- TCCCCCCGGGGGTTTCATAAGACCGCTGATGA Dn-R (SEQ ID NO: 56) Als2- TGCCGATACAAGAACAACAGC Pc-F (SEQ ID NO: 57) Als2- ATTACTTTCATAGCCCTCACTCCTCCACGGGT Pc-R TCGCTACCTGCATTA (SEQ ID NO: 58) Als2- GGAGGAGTGAGGGCTATGAAAGTAATTTTTTT ldh-F TTC (SEQ ID NO: 59) Als4- TGCCCCGGCCTGCATGCACGTCGACACGGGTA up-R CATTTGAAGGATCTTG (SEQ ID NO: 60) Als4- TGTCGACGTGCATGCAGGCC P43-F (SEQ ID NO: 61) Als4- ATTACTTTCATAGCCCTCACTCCTCCTATAAT P43-R GGTACCGCTATCAC (SEQ ID NO: 62) Als6- TTTCTTCCTTAAATTTGTACATTTTCCGGGTA up-R CATTTGAAGGATCTTG (SEQ ID NO: 63) Als6- GAAAATGTACAAATTTAAGG Pldh-F (SEQ ID NO: 64) Als6- ATTACTTTCATAGCCCTCACTCCTCCGACTCAT Pldh-R CATTCCTTTGCCG (SEQ ID NO: 65) AlsA- TTTGTGCTATAAACGGCGAGTTTCATAGCCCTC up-R ACTCCTCCATTTTCA (SEQ ID NO: 66) AlsA- ATGAAACTCGCCGTTTATAGC ldhA-F (SEQ ID NO: 67) AlsA- CCCCTTTTCAAAGAGGGCTTTTTCATTTAAACC ldhA-R AGTTCGTTCGGGCA (SEQ ID NO: 68) Notes: “-F” represents forward primer; “-R” represents reverse primer; and underlines represent enzyme digestion sites.

3) Construction of Pyruvate Formate-Lyase (PflA) Gene Knockout Plasmids in Pathway of Formic Acid

By using ATCC 14580 genome DNA as a template and primer pairs pflA1-F/pflA1-R and pflA2-F/pflA2-R, PCR amplification was respectively performed to upstream and downstream homologous arms of a pyruvate formate-lyase gene, then the upstream and downstream homologous arms were subjected to double enzyme digestion with EcoRI/XhoI and XhoI/BamHIx, simultaneously the plasmid pKVM1 was subjected to double enzyme digestion with BamHI/NcoI, the vector and segments after enzyme digestion were linked by using T4 DNA ligase and then transformed into E. coli S17-1, and after correctness was verified through DNA sequencing, a positive clone plasmid was named as pKVMΔpflA.

Herein, primer sequences are as follows (5′-3′):

pflA1-F: (SEQ ID NO: 73) CCGGAATTCATGGAACAATGGAAAGGT pflA1-R: (SEQ ID NO: 74) TCGCTCGAGTGGAAATGTCAAACCCATAG pflA2-F: (SEQ ID NO: 75) CCGCTCGAGGACGATGGCCACTGGGATC pflA2-R: (SEQ ID NO: 76) ATCGGATCCCTACATCGATTCATGGAAGG

4) Construction of Knockout Plasmids for Ethanol Dehydrogenase (AdhB) Gene in Pathway of Ethanol

By using ATCC 14580 genome DNA as a template and primer pairs adhB-up-F/adhB-up-R and adhB-dn-F/adhB-dn-R, PCR amplification was respectively performed to upstream and downstream homologous arms of a pyruvate formate-lyase gene, then the upstream and downstream homologous arms were subjected to double enzyme digestion with BamHI/XhoI and XhoI/NcoI, simultaneously the plasmid pKVM1 was subjected to double enzyme digestion with BamHI/NcoI, the vector and segments after enzyme digestion were linked by using T4 DNA ligase and then transformed into E. coli S17-1, and after correctness was verified through DNA sequencing, a positive clone plasmid was named as pKVMΔadhB.

Herein, primer sequences are as follows (5′-3′):

adhB-up-F: (SEQ ID NO: 77) TACGGGATCCGAACGGGAATCGGCAAAGGGATT adhB-up-R: (SEQ ID NO: 78) TCCGCTCGAGCGATGATAAAGGCTGCCGAGCTA adhB-dn-F: (SEQ ID NO: 79) ACCGCTCGAGTCGTCACACTCCCATTATCG adhB-dn-R: (SEQ ID NO: 80) CATGCCATGGCGTCGTATTTGCCGTCAGCT

5) Construction of Knockout Plasmids for Acetokinase (Ack) Gene in Pathway of Acetic Acid

By using ATCC 14580 genome DNA as a template and primer pairs ack-up-F/ack-up-R and ack-dn-F/ack-dn-R, PCR amplification was respectively performed to upstream and downstream homologous arms of a pyruvate formate-lyase gene, then the upstream and downstream homologous arms were subjected to double enzyme digestion with BamHI/XhoI and XhoI/NcoI, simultaneously the plasmid pKVM1 was subjected to double enzyme digestion with BamHI/NcoI, the vector and segments after enzyme digestion were linked by using T4 DNA ligase and then transformed into E. coli S17-1, and after correctness was verified through DNA sequencing, a positive clone plasmid was named as pKVMΔack.

Herein, primer sequences are as follows (5′-3′):

ack-up-F: (SEQ ID NO: 81) TACGGGATCCGAAGGCTTTCCGGCCTTACT ack-up-R: (SEQ ID NO: 82) TCCGCTCGAGCATCGTCATTCCGACGAATG ack-dn-F:  (SEQ ID NO: 83) ACCGCTCGAGGAGCTTCCAGCATTGATTGC ack-dn-R: (SEQ ID NO: 84) CATGCCATGGATGCGGTCATCTGCGATCTT

2. Use of the Constructed Vector for Reconstructing Heat-Resistant Bacillus Licheniformis.

1) Steps for Knocking Out Gene of Heat-Resistant Bacillus Licheniformis:

(1) E. coli S17-1 containing knockout plasmids and Bacillus licheniformis MW3 were respectively cultured to OD_(600 nm)≈1.2 in LB culture mediums, centrifugal separation was performed for 5 min at 6000 rpm, washing with 0.9% normal saline was performed twice, the two bacterium were mixed, resuspended and centrifugalized, then the bacterium was resuspended by using LB culture mediums, and dropped onto an LB plate, with overnight incubation at 30° C., then the cells were collected by using preheated (30° C.) LB, coated onto an LB solid plate (erythrocin and polymyxin B), and cultured at 30° C.;

(2) Transformants were picked, transferred into LB culture mediums (erythrocin) and cultured at 30° C., the transformants after culture were transferred onto an LB plate (erythrocin and X-Gal) in dilution series and cultured overnight at 42° C., and blue bacterial colonies were correct transformants.

(3) Transformants were picked, transferred into LB culture mediums, and subjected to resistant-free culture at 30° C., cultivated for two generations, diluted and coated onto an LB plate (X-Gal), and white transformants were picked and molecular verification was performed.

2) Preparation Process of Knockout Strain for Heat-Resistant Bacillus Licheniformis

A host strain selected and used for genetic operation was a mutant strain MW3 in which a restrictive modification system (ΔhsdR1, ΔhsdR2) was knocked out, the mutant strain can perform transformation of exogenous DNA more efficiently, and simultaneously the capabilities of growth and secretion of protein are the same as that of wild ATCC 14580 and are not influenced by knockout. Preparation of a heat-resistant Bacillus licheniformis MW3 knockout strain was as follow:

(1) Preparation of host strain BL2 in which L-lactate dehydrogenase (Ldh) gene was knockout: biparental combination was performed to the strain MW3 and E. coli S17-1 containing knockout plasmids pKVMΔldh by using the above-mentioned genetic operation method, then transformants were obtained respectively through single crossover and double crossover, then a genome was extracted, and verified through PCR, and positive strains were picked and a next experiment was performed.

(2) Preparation of host strain BN11 in which L-lactate dehydrogenase (Ldh) gene, acetolactate synthase (alsS) gene and acetolactate decarboxylase (alsD) gene in pathway of 2,3-butanediol were knockout and promoter P_(als) was reserved: biparental combination was performed to the strain BL2 and E. coli S17-1 containing knockout plasmids pKVMN1 by using the above-mentioned genetic operation method, then transformants were obtained respectively through single crossover and double crossover, then a genome was extracted and verified through PCR, and positive strains were picked and a next experiment was performed.

(3) Preparation of host strain BN22 in which L-lactate dehydrogenase (Ldh) gene, acetolactate synthase (alsS) gene and acetolactate decarboxylase (alsD) gene in pathway of 2,3-butanediol were knockout and promoter P_(als) was replaced with promoter P_(c): biparental combination was performed to the strain BL2 and E. coli S17-1 containing knockout plasmids pKVMN2 by using the above-mentioned genetic operation method, then transformants were obtained respectively through single crossover and double crossover, then a genome was extracted and verified through PCR, and positive strains were picked and a next experiment was performed.

(4) Preparation of host strain BN44 in which L-lactate dehydrogenase (Ldh) gene, acetolactate synthase (alsS) gene and acetolactate decarboxylase (alsD) gene in pathway of 2,3-butanediol were knockout and promoter P_(als) was replaced with promoter P₄₃: biparental combination was performed to the strain BL2 and E. coli S17-1 containing knockout plasmids pKVMN4 by using the above-mentioned genetic operation method, then transformants were obtained respectively through single crossover and double crossover, then a genome was extracted and verified through PCR, and positive strains were picked and a next experiment was performed.

(5) Preparation of host strain BN66 in which L-lactate dehydrogenase (Ldh) gene, acetolactate synthase (alsS) gene and acetolactate decarboxylase (alsD) gene in pathway of 2,3-butanediol were knockout and promoter P_(als) was replaced with promoter P_(ldh): biparental combination was performed to the strain BL2 and E. coli S17-1 containing knockout plasmids pKVMN6 by using the above-mentioned genetic operation method, then transformants were obtained respectively through single crossover and double crossover, then a genome was extracted and verified through PCR, and positive strains were picked and a next experiment was performed.

(6) Preparation of host strain BA11 in which thermophilic D-lactate dehydrogenase Ldh_(Ti) in BN11 was replaced with mesophilic D-lactate dehydrogenase: biparental combination was performed to the strain BN11 and E. coli S17-1 containing knockout plasmids pKVMA1 by using the above-mentioned genetic operation method, then transformants were obtained respectively through single crossover and double crossover, then a genome was extracted and verified through PCR, and positive strains were picked and a next experiment was performed.

(7) Preparation of host strain BN12 in which pyruvate formate-lyase (PflA) gene was knocked out starting from strain BN11: biparental combination was performed to the strain BN11 and E. coli S17-1 containing knockout plasmids pKVMΔpflA by using the above-mentioned genetic operation method, then transformants were obtained respectively through single crossover and double crossover, then a genome was extracted and verified through PCR, and positive strains were picked and a next experiment was performed.

(8) Preparation of host strain BN13 in which ethanol dehydrogenase (AdhB) gene was knocked out starting from strain BN12: biparental combination was performed to the strain BN11 and E. coli S17-1 containing knockout plasmids pKVMΔadhB by using the above-mentioned genetic operation method, then transformants were obtained respectively through single crossover and double crossover, then a genome was extracted and verified through PCR, and positive strains were picked and a next experiment was performed.

(9) Preparation of host strain BN13 in which acetokinase (Ack) gene was knocked out starting from strain BN13: biparental combination was performed to the strain BN11 and E. coli S17-1 containing knockout plasmids pKVMΔack by using the above-mentioned genetic operation method, then transformants were obtained respectively through single crossover and double crossover, then a genome was extracted and verified through PCR, and positive strains were selected and a next experiment was performed.

Embodiment 4: Fermentation of Original Strain, Ldh-Knocked-Out Strain and Strains Containing Different Promoters and Gene Ldh_(Ti)

This embodiment was performed in a 5 L full-automatic fermentation tank, and components of various used culture mediums were as follows:

Each liter of slant culture mediums contained: 30-50 g of glucose, 5-10 g of yeast powder, 2-8 g of peptone, 30 g of calcium carbonate, 15-25 g of agar powder and balance of water. pH of the slant culture mediums was 7.0. Sterilization was performed for 15 min at 115° C.

Each liter of seed culture mediums contained: 40-120 g of glucose, 5-10 g of yeast powder, 2-8 g of peptone, 50 g of calcium carbonate and balance of water. pH of the seed culture mediums was 6.0-8.0. Sterilization was performed for 15 min at 115° C.

Each liter of fermentation culture mediums contained: 120 g of glucose, 5-10 g of yeast powder, 2-8 g of peptone, 50 g of calcium carbonate and balance of water. pH of the fermentation culture mediums was 6.5-7.5. Sterilization was performed for 15 min at 115° C.

The method for producing D-lactic acid through fermentation in this embodiment comprised the following steps:

(1) Slant culture: the strain was inoculated into the slant culture mediums and cultured for 24 h at 50° C.

(2) Seed culture: the strains cultured in step (1) were inoculated into a 40 mL triangular flask containing 40 mL of seed culture mediums under aseptic conditions by using an inoculating loop for two times, and subjected to static culture for 24 h at 50° C. to obtain seed culture solution 1; and 10 mL seed culture solution 1 was inoculated into a 500 mL triangular flask containing 100 mL seed culture medium under aseptic conditions, and subjected to static culture for 24 h at 50° C. to obtain seed culture solution 2.

(3) Fermentation culture: 300 mL seed culture solution 2 prepared in step (2) was inoculated into a fermentation tank containing 2.7 L fermentation culture mediums under aseptic conditions, and subjected to culture at 50° C. under stirring at 70 rpm, sampling was performed once every 3 h, and fermentation was ended after 12 h.

After fermentation was ended, supernatant of the fermentation liquid was taken and analyzed for concentrations of D-lactic acid, byproducts and total reducing sugar through HPLC, and sugar-acid conversion rate was calculated (Table 4), wherein conversion rate (%)=yield of lactic acid (g/L)/consumption of glucose (g/L)*100%. Herein, through HPLC for detecting D-lactic acid and L-lactic acid, it can be seen that the optical purity of D-lactic acid produced by BN11 can reach 99.9% (FIG. 6).

TABLE 4 Glucose Consumption and Product Production for Different Strains Product (g/L) Glucose L- D- Conversion Consumption 2, 3- lactic lactic Formic Acetic Rate Strain (g/L) butanediol Acid Acid Acid Acid Ethanol (%) MW3 42.9 11.4 7.6 — 3.7 1.9 3.0 17.7  BL2 52.1 18.4 — — 4.8 2.7 3.3 — BN11 85.1 — — 76.8  2.1 2.7 1.8 90.2  BN22 17.5 — —  0.52 4.1 3.2 2.9 3.0 BN44 55.0 — — 35.9  4.5 3.8 5.0 65.3  BN66 45.3 — — 8.1 12.3  4.7 5.4 17.9  “—” represents that the amount of product is smaller than 0.01 g/L.

Since the original strain Bacillus licheniformis ATCC 14580 adopted in this embodiment can produces 2,3-butanediol with a high yield, i.e., it indicates that the activity of this pathway is high, and Pals is a promoter of a first acetolactate synthase gene in a gene cluster for synthesis of 2,3-butanediol, this is possibly the reason why the gene engineering strain BN11 (adopting the promoter) presents a good conversion rate.

Embodiment 5: Fermentation of an Optimal Strain in Embodiment 4 after Blocking of Byproduct Production

This embodiment was performed in a 5 L full-automatic fermentation tank, the used strains included BN11, BN12 (with a pathway for producing byproduct formic acid blocked), BN13 (with pathways for producing byproducts formic acid and ethanol blocked) and BN14 (with pathways for producing byproducts formic acid, ethanol and acetic acid blocked), and components of various used culture mediums were as follows:

Slant culture mediums, seed culture mediums and fermentation culture mediums were the same as that in Embodiment 4.

A method for producing D-lactic acid through fermentation was the same as that in Embodiment 4.

After fermentation was ended, supernatant of the fermentation liquid was taken and analyzed for concentrations of D-lactic acid, byproducts and total reducing sugar through HPLC, and sugar-acid conversion rate was calculated (Table 5), wherein conversion rate (%)=yield of lactic acid (g/L)/consumption of glucose (g/L)*100%. Herein, through HPLC for detecting D-lactic acid and L-lactic acid, it can be seen that the optical purity of D-lactic acid produced by BN11 can reach 99.9%.

TABLE 5 Glucose Consumption and Product Production of Different Strains with Pathways for Producing Byproducts Blocked Product (g/L) Glucose L- D- Conversion Consumption 2, 3- lactic lactic Formic Acetic Rate Strain (g/L) butanediol Acid Acid Acid Acid Ethanol (%) BN11 85.1 — — 76.8 2.1 2.7 1.8 90.2 BN12 85.2 — — 77.5 1.0 2.8 1.7 91.0 BN13 85.1 — — 79.0 1.1 2.7 0.1 92.8 BN14 85.3 — — 80.5 1.0 1.2 0.1 94.4 “—” represents that the amount of product is smaller than 0.01 g/L.

As shown by results, after generation of 2,3-butanediol and L-lactic acid is blocked, by further blocking pathways for producing other byproducts, the conversion rate from glucose to D-lactic acid can be improved. In addition, the more the blocked pathways are, the higher the conversion rate of D-lactic acid is.

Embodiment 6: Optimization of Fermentation Conditions of Optimal Strain in Embodiment 4

This embodiment was performed in a 5 L full-automatic fermentation tank, the used strain was BN11, and components of various used culture mediums were as follows:

Slant culture mediums and the seed culture mediums were the same as that in Embodiment 4.

Each liter of fermentation culture mediums contained: 1) 90 g of glucose, 5-10 g of yeast powder, 2-8 g of peptone and balance of water when the optimal fermentation pH was determined, wherein pH was respectively regulated to 6.0, 6.5, 7.0, 7.5 and 8.0; and 2) 5-10 g of yeast powder, 2-8 g of peptone, 60 g, 87 g, 122 g, 148 g, 180 g and 202 g of glucose and balance of water in order to determine capabilities of the strain in producing D-lactic acid in culture mediums with different concentrations of glucose.

The method for producing D-lactic acid through fermentation in this embodiment comprised the following steps:

(1) Slant culture: same as that in Embodiment 4.

(2) Seed culture: same as that in Embodiment 4.

(3) Fermentation culture: 300 mL of seed culture solution 2 prepared in step (2) was inoculated into a fermentation tank containing 2.7 L of different fermentation culture mediums under aseptic conditions, and cultured at 50° C. under stirring at 70 rpm, sampling was performed once every 3 h, and fermentation was ended after 12 h.

After fermentation was ended, supernatant of the fermentation liquid was taken and analyzed for concentrations of D-lactic acid, byproducts and total reducing sugar through HPLC, and sugar-acid conversion rate was calculated.

As proved by experiment results, the optimal fermentation pH for the experiment strain BN11 at 50° C. is 7.0; and at the pH value, the consumption speed of glucose was the largest, the sugar-acid conversion rate is the highest and the amount of the byproducts is the least (Table 6). From FIG. 7, it can be seen that, when the concentration of glucose does not exceed 180 g/L, the speed at which the strain BN11 consumes glucose or produces D-lactic is not obviously inhibited; and when the concentration of glucose is higher than 180 g/L such 202 g/L, the consumption of glucose is obviously inhibited and the production speed of D-lactic acid is reduced accordingly.

TABLE 6 Influence of pH on Production of D-lactic Acid by BN11 Product (g/L) Cell Glucose D- Conversion Density Consumption lactic Formic Acetic Rate pH (OD_(600nm)) (g/L) Acid Acid Acid Ethanol (%) 6.0 3.3 19.8 16.9 —  0.90  0.51 85.3 6.5 9.1 55.4 49.1  0.30 2.6  0.72 88.6 7.0 10.5  86.1 77.8 2.6 3.6 1.8 90.4 7.5 10.2  83.4 67.6 6.5 5.4 1.0 81.1 8.0 9.3 71.7 55.2 5.9 4.6 1.6 77.0 “—” represents that the amount of product is smaller than 0.01 g/L.

Embodiment 7: Production of D-Lactic Acid by Using Optimal Strain in Embodiment 4 through Batch Fermentation and Fed (Sugar)-Batch Fermentation

This embodiment was performed in a 5 L full-automatic fermentation tank, the used strain was BN11, and components of various used culture mediums were as follows:

The slant culture mediums and the seed culture mediums were the same as that in Embodiment 4.

Each liter of fermentation culture mediums contained: 1) 180 g of glucose, 5-10 g of yeast powder, 2-8 g of peptone and balance of water when D-lactic acid was produced through batch fermentation; and 2) 40-70 g of glucose, 5-10 g of yeast powder, 2-8 g of peptone and balance of water when D-lactic acid was produced through fed (sugar)-batch fermentation.

The method for producing D-lactic acid through fermentation in this embodiment comprised the following steps:

(1) Slant culture: same as that in Embodiment 4.

(2) Seed culture: same as that in Embodiment 4.

(3) Fermentation culture: 300 mL of seed culture solution 2 prepared in step (2) was inoculated into fermentation tanks respectively containing 2.7 L of batch fermentation culture mediums and fed (sugar)-batch fermentation culture mediums under aseptic conditions, and cultured at 50° C. under stirring at 70 rpm, sampling was performed once every 2-5 h, and the amount of residual sugar in the fermentation liquid was determined. During batch fermentation, when glucose was fully consumed or the consumption speed tended to 0 in the fermentation process, fermentation was ended; and during fed (sugar)-batch fermentation, when the concentration of glucose decreased to 10-20 g/L, glucose was fed in batch to enable the concentration of glucose to reach 50-70 g/L, and totally the glucose was supplemented for 2-5 times. When the consumption speed of glucose in the fermentation process tended to 0, fermentation was ended.

After fermentation is ended, supernatant of the fermentation liquid was taken and analyzed for concentrations of D-lactic acid, byproducts and total reducing sugar through HPLC, and sugar-acid conversion rate and production speed were calculated.

From FIG. 8A, it can be seen that, when batch fermentation is performed by using the experiment strain BN11 at 50° C., glucose is fully consumed and fermentation is ended after 40 h. The concentration of the produced D-lactic acid is 167.7 g/L, the concentration of the byproduct ethanol is 5.8 g/L, the concentration of formic acid is 1.1 g/L and the concentration of acetic acid is 3.6 g/L. The sugar-acid conversion rate and the production speed are respectively 93.0% and 4.2 g/[L·h].

From FIG. 8B, it can be seen that, when fed (sugar)-batch fermentation is performed by using the strain BN11 at 50° C., totally glucose is fed for four times and fermentation is ended after 70 h, wherein in the first 38 h, 173.2 g/L D-lactic acid is produced and the production speed is 4.6 g/[L·h]; and from 38 h to 70 h, the concentration of D-lactic acid increases from 173.2 g/L to 226.6 g/L, and the production speed is 1.7 g/[L·h]. After fermentation is ended, totally 242.1 g/L glucose is consumed, the average production speed is 3.2 g/[L·h] and the sugar-acid conversion rate is 93.6%. The final concentration of the byproduct formic acid is 0.56 g/L, and the final concentrations of acetic acid and ethanol are respectively 5.5 g/L and 4.3 g/L.

Embodiment 8: Production of D-Lactic Acid by Using Strain in which Thermophilic Ldh_(Ti) is Replaced with Mesophilic D-lactate Dehydrogenase (Ldha) through Batch Fermentation and Fed (Sugar)-Batch Fermentation

This embodiment was performed in a 5 L full-automatic fermentation tank, the used strain was BA11, and components of various used culture mediums were as follows:

Slant culture mediums and seed culture mediums were the same as that in Embodiment 4.

The content contained in each liter of fermentation culture mediums was the same as that in Embodiment 7.

The method for producing D-lactic acid through fermentation in this embodiment comprised the following steps:

(1) Slant culture: same as that in Embodiment 4.

(2) Seed culture: same as that in Embodiment 4.

(3) Fermentation culture: same as that in Embodiment 7.

After fermentation was ended, supernatant of the fermentation liquid was taken and analyzed for concentrations of D-lactic acid, byproducts and total reducing sugar through HPLC, and sugar-acid conversion rate and production speed were calculated.

From FIG. 9A, it can be seen that, when batch fermentation is performed by using the experiment strain BA11 at 50° C., fermentation is ended after 40 h and the concentration of remaining residual sugar is 57.1 g/L. The concentration of the finally produced D-lactic acid is 100.8 g/L, the concentration of the byproduct ethanol is 9.5 g/L, the concentration of formic acid is 3.9 g/L and the concentration of acetic acid is 4.5 g/L. The sugar-acid conversion rate and the production speed are respectively 82.4% and 2.5 g/[L·h].

From FIG. 9B, it can be seen that, when fed (sugar)-batch fermentation is performed by using the strain BA11 at 50° C., totally glucose is fed for three times and fermentation is ended after 70 h. Finally, totally 196.9 g/L glucose is consumed, the produced D-lactic acid is 168.6 g/L, the average production speed is 2.4 g/[L·h] and the sugar-acid conversion rate is 85.6%. The final concentrations of the byproducts formic acid and ethanol are 1.0 g/L, and the final concentration of acetic acid is 5.5 g/L.

Embodiment 9: Production of D-Lactic Acid by Using Optimal Strain in Embodiment 4 Through Fed-Batch Fermentation by Using Xylose

This embodiment was performed in a 5 L full-automatic fermentation tank, the used strain was BN11, and components of various used culture mediums were as follows:

Each liter of slant culture mediums contained: 30-50 g of xylose, 5-10 g of yeast powder, 2-8 g of peptone, 30 g of calcium carbonate, 15-25 g of agar powder and balance of water. pH of the slant culture mediums was 7.0. Sterilization was performed for 15 min at 115° C.

Each liter of seed culture mediums contained: 40-70 g of xylose, 5-10 g of yeast powder, 2-8 g of peptone, 50 g of calcium carbonate and balance of water. pH of the seed culture mediums was 6.0-8.0. Sterilization was performed for 15 min at 115° C.

Each liter of fermentation culture mediums contained: 40-60 g of xylose, 5-10 g of yeast powder, 2-8 g of peptone and balance of water. pH of the fermentation culture mediums was 6.5-7.5. Sterilization was performed for 15 min at 115° C.

The method for producing D-lactic acid through fermentation in this embodiment comprised the following steps:

(1) Slant culture: same as that in Embodiment 4.

(2) Seed culture: same as that in Embodiment 4.

(3) Fermentation culture: 300 mL of seed culture solution prepared in step (2) was inoculated into a fermentation tank containing 2.7 L of fermentation culture mediums under aseptic conditions, and cultured at 50° C. under stirring at 70 rpm, sampling was performed once every 2-5 h, and the amount of residual sugar in the fermentation liquid was determined. When the concentration of xylose decreased to 10-20 g/L, xylose was fed in batch to enable the concentration of xylose to reach 40-60 g/L, and totally the xylose was supplemented for 2-5 times. When the consumption speed of xylose in the fermentation process tended to 0, fermentation was ended.

After fermentation was ended, supernatant of the fermentation liquid was taken and analyzed for concentrations of D-lactic acid, byproducts and total reducing sugar through HPLC, and sugar-acid conversion rate and production speed were calculated.

From FIG. 10, it can be see that, when fed-batch fermentation is performed by using the strain BN11 and xylose as a carbon source at 50° C., totally xylose is supplemented once and fermentation is ended after 70 h. Finally, totally 114.7 g/L xylose is consumed, the produced D-lactic acid is 72.1 g/L, and the average production speed is 1.0 g/[L·h]. The final concentration of the byproduct formic acid is 23.0 g/L, and the final concentrations of acetic acid and ethanol are respectively 7.2 g/L and 8 g/L.

Embodiment 10: Production of D-Lactic Acid by Using Optimal Strain in Embodiment 4 Through Fed (Sugar)-Batch Fermentation by Using Low-Cost Culture Mediums

This embodiment was performed in a 5 L full-automatic fermentation tank, the used strain was BN11, and components of various used culture mediums were as follows:

Slant culture mediums and seed culture mediums were the same as that in Embodiment 4.

Each liter of fermentation culture mediums contained: 1) 40-70 g of glucose, 1-5 g of yeast powder, 0.25-1.0 g/L potassium dihydrogen phosphate, 0.25-1.0 g/L dipotassium phosphate, 1.5-10.0 g/L ammonium sulfate, 1.2-5.0 g/L diammonium hydrogen phosphate, 0.15-0.8 g/L zinc sulfate, 1-10 g/L dried corn steep powder and balance of water. Sterilization was performed for 15 min at 115° C.

The method for producing D-lactic acid through fermentation in this embodiment comprised the following steps:

(1) Slant culture: same as that in Embodiment 4.

(2) Seed culture: same as that in Embodiment 4.

(3) Fermentation culture: 600 mL of seed culture solution 2 prepared in step (2) was inoculated into a fermentation tank containing 2.4 L of low-cost fermentation culture mediums under aseptic conditions, and cultured at 50° C. under stirring at 70 rpm, sampling was performed once every 2-5 h, and the amount of residual sugar in the fermentation liquid was determined. When the concentration of glucose decreased to 10-20 g/L, glucose was fed in batch to enable the concentration of glucose to reach 50-70 g/L, and totally the glucose was supplemented for 2-5 times. When the consumption speed of glucose in the fermentation process tended to 0, fermentation was ended.

After fermentation was ended, supernatant of the fermentation liquid was taken and analyzed for concentrations of D-lactic acid, byproducts and total reducing sugar through HPLC, and sugar-acid conversion rate and production speed were calculated.

From FIG. 11, it can be seen that, when fed (sugar)-batch fermentation is performed by using the strain BN11 and low-cost culture mediums at 50° C., totally glucose is fed in batch for four times and fermentation is ended after 90 h. Totally 190.9 g/L glucose is consumed, the produced D-lactic acid is 175.7 g/L, the average production speed is 2.0 g/[L·h] and the sugar-acid conversion rate is 92.0%. The final concentration of the byproduct acetic acid is 7.5 g/L, and the final concentrations of formic acid and ethanol are respectively 0.62 g/L and 1.6 g/L.

The preferred embodiments of the present invention are described above. It shall be understood that one skilled in the art may make various modifications and variations according to the concept of the present invention without contributing any inventive labor. Therefore, all technical solutions obtained by one skilled in the art according to the concept of the present invention on the basis of the prior art in combination with logical analysis, reasoning or limited experiments shall be included in the protective scope determined by the claims. 

The invention claimed is:
 1. A nucleotide sequence, wherein the nucleotide sequence encodes a D-lactate dehydrogenase comprising a substitution, deletion, insertion, addition of one or more amino acid residues, wherein said D-lactate dehydrogenase comprises an amino acid sequence having at least 80% and less than 100% sequence identity to SEQ ID No. 1, and wherein said D-lactate dehydrogenase has D-lactate dehydrogenase activity.
 2. The nucleotide sequence according to claim 1, wherein the nucleotide sequence is one of the following nucleotide sequences: 1) a nucleotide sequence of SEQ ID No. 2; 2) a nucleotide sequence of SEQ ID No. 3; 3) a nucleotide sequence having more than 80% of homology with the nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 3; and 4) a nucleotide sequence hybridized with a complementary chain of the nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 3 under a high-stringency condition.
 3. A gene engineering strain, wherein the gene engineering strain has a D-lactate dehydrogenase, wherein said D-lactate dehydrogenase comprises a substitution, deletion, insertion, addition of one or more amino acid residues, wherein said D-lactate dehydrogenase comprises an amino acid sequence having at least 80% and less than 100% sequence identity to SEQ ID No. 1, and wherein said D-lactate dehydrogenase has D-lactate dehydrogenase activity.
 4. The gene engineering strain according to claim 3, wherein a nucleotide sequence encoding the D-lactate dehydrogenase is one of the following nucleotide sequences: 1) a nucleotide sequence of SEQ ID No. 2; 2) a nucleotide sequence of SEQ ID No. 3; 3) a nucleotide sequence having more than 80% of homology with the nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 3; and 4) a nucleotide sequence hybridized with a complementary chain of the nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 3 under a high-stringency condition.
 5. The gene engineering strain according to claim 3, wherein in the gene engineering strain, a pathway for synthesizing L-lactic acid by pyruvic acid is blocked and a pathway for synthesizing 2,3-butanediol by pyruvic acid is blocked.
 6. The gene engineering strain according to claim 5, wherein the pathway for synthesizing L-lactic acid by pyruvic acid is blocked by deactivating or deleting an L-lactate dehydrogenase gene; and the pathway for synthesizing 2,3-butanediol by pyruvic acid is blocked by deactivating or deleting one or both of an acetolactate synthase gene and an acetolactate decarboxylase gene.
 7. The gene engineering strain according to claim 5, wherein in the gene engineering strain, one or more of a pathway for synthesizing formic acid by pyruvic acid, a pathway for synthesizing acetic acid by pyruvic acid and a pathway for synthesizing ethanol by pyruvic acid are blocked.
 8. The gene engineering strain according to claim 7, wherein the pathway for synthesizing formic acid by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate formate-lyase gene and a pyruvate formate-lyase activating enzyme gene; the pathway for synthesizing acetic acid by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate dehydrogenase gene and an acetokinase gene; and the pathway for synthesizing ethanol by pyruvic acid is blocked by deactivating or deleting one or both of a pyruvate dehydrogenase gene and an ethanol dehydrogenase gene.
 9. The gene engineering strain according to claim 5, wherein an original D-lactate dehydrogenase gene in the gene engineering strain is deactivated or deleted.
 10. The gene engineering strain according to claim 5, wherein an original strain of the gene engineering strain is thermophilus.
 11. The gene engineering strain according to claim 5, wherein an original strain of the gene engineering strain is bacillus.
 12. The gene engineering strain according to claim 11, wherein the bacillus is selected from a group consisting of Bacillus licheniformis, Bacillus coagulans, B. subtilis, Bacillus amyloliquefaciens, Bacillus pumilus, Bacillus circulans and Aneurinibacillus aneurinilyticus.
 13. The gene engineering strain according to claim 10, wherein a restrictive modification system in the original strain is deactivated or knocked out.
 14. The gene engineering strain according to claim 13, wherein the original strain is a ΔhsdR1ΔhsdR2 double-mutant strain MW3 of Bacillus licheniformis ATCC
 14580. 15. The gene engineering strain according to claim 10, wherein a promoter of a coding gene of the D-lactate dehydrogenase is reconstructed into a constitutive promoter.
 16. The gene engineering strain according to claim 15, wherein the constitutive promoter is a promoter P_(als) of α-acetolactate synthase of Bacillus licheniformis ATCC 14580, a P_(c) promoter in plasmid pMMPc, a P₄₃ promoter in B. subtilis or a promoter P_(ldh) of L-lactate dehydrogenase of Bacillus licheniformis ATCC
 14580. 17. The gene engineering strain according to claim 15, wherein the constitutive promoter is a promoter P_(als) of α-acetolactate synthase of Bacillus licheniformis ATCC
 14580. 18. The gene engineering strain according to claim 3, wherein the gene engineering strain is Bacillus licheniformis BN11, the preservation number of which is CCTCC NO: M2016026 and which was preserved in China Center for Type Culture Collection on Jan. 8,
 2016. 19. A method for preparing the gene engineering strain according to claim 3, wherein the method comprises: deactivating or deleting an L-lactate dehydrogenase gene in an original strain; and introducing the nucleotide sequence of claim
 1. 20. The method according to claim 19, wherein the nucleotide sequence encoding the D-lactate dehydrogenase is one of the following nucleotide sequences: 1) a nucleotide sequence of SEQ ID No. 2; 2) a nucleotide sequence of SEQ ID No. 3; 3) a nucleotide sequence having more than 80% of homology with the nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 3; and 4) a nucleotide sequence hybridized with a complementary chain of the nucleotide sequence of SEQ ID No. 2 or SEQ ID No. 3 under a high-stringency condition.
 21. The method according to claim 19, wherein the method further comprises: deactivating or deleting an original D-lactate dehydrogenase gene in the original strain; and blocking a pathway for synthesizing 2,3-butanediol by pyruvic acid.
 22. The method according to claim 21, wherein the method further comprises: reconstructing a promoter of the introduced nucleotide sequence into a constitutive promoter.
 23. The method according to claim 22, wherein the original strain is a ΔhsdR1ΔhsdR2 double-mutant strain MW3 of Bacillus licheniformis ATCC 14580; and α-acetolactate decarboxylase (alsD) and acetolactate synthase (alsS) genes are replaced with the nucleotide sequence encoding the D-lactate dehydrogenase. 